Endothelium-specific depletion of LRP1 improves glucose homeostasis through inducing osteocalcin.

The vascular endothelium is present within metabolic organs and actively regulates energy metabolism. Here we show osteocalcin, recognized as a bone-secreted metabolic hormone, is expressed in mouse primary endothelial cells isolated from heart, lung and liver. In human osteocalcin promoter-driven green fluorescent protein transgenic mice, green fluorescent protein signals are enriched in endothelial cells lining aorta, small vessels and capillaries and abundant in aorta, skeletal muscle and eye of adult mice. The depletion of lipoprotein receptor-related protein 1 induces osteocalcin through a Forkhead box O -dependent pathway in endothelial cells. Whereas depletion of osteocalcin abolishes the glucose-lowering effect of low-density lipoprotein receptor-related protein 1 depletion, osteocalcin treatment normalizes hyperglycemia in multiple mouse models. Mechanistically, osteocalcin receptor-G protein-coupled receptor family C group 6 member A and insulin-like-growth-factor-1 receptor are in the same complex with osteocalcin and required for osteocalcin-promoted insulin signaling pathway. Therefore, our results reveal an endocrine/paracrine role of endothelial cells in regulating insulin sensitivity, which may have therapeutic implications in treating diabetes and insulin resistance through manipulating vascular endothelium.

Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

Brain endothelial LRP1 maintains blood-brain barrier integrity.

The entry of blood-borne molecules into the brain is restricted by the blood-brain barrier (BBB). Various physical, transport and immune properties tightly regulate molecule movement between the blood and the brain to maintain brain homeostasis. A recent study utilizing a pan-endothelial, constitutive Tie2-Cre showed that paracellular passage of blood proteins into the brain is governed by endocytic and cell signaling protein low-density lipoprotein receptor-related protein 1 (LRP1). Taking advantage of conditional Slco1c1-CreERT2 specific to CNS endothelial cells and choroid plexus epithelial cells we now supplement previous results and show that brain endothelial Lrp1 ablation results in protease-mediated tight junction degradation, P-glycoprotein (P-gp) reduction and a loss of BBB integrity.

Hepatic LDL receptor-related protein-1 deficiency alters mitochondrial dynamics through phosphatidylinositol 4,5-bisphosphate reduction.

The LDL receptor-related protein 1 (LRP1) is a multifunctional transmembrane protein with endocytosis and signal transduction functions. Previous studies have shown that hepatic LRP1 deficiency exacerbates diet-induced steatohepatitis and insulin resistance via mechanisms related to increased lysosome and mitochondria permeability and dysfunction. The current study examined the impact of LRP1 deficiency on mitochondrial function in the liver. Hepatocytes isolated from liver-specific LRP1 knockout (hLrp1-/-) mice showed reduced oxygen consumption compared with control mouse hepatocytes. The mitochondria in hLrp1-/- mouse livers have an abnormal morphology and their membranes contain significantly less anionic phospholipids, including lower levels of phosphatidylethanolamine and cardiolipin that increase mitochondrial fission and impair fusion. Additional studies showed that LRP1 complexes with phosphatidylinositol 4-phosphate 5-kinase like protein-1 (PIP5KL1) and phosphatidylinositol 4-phosphate 5-kinase-1ß (PIP5K1ß). The absence of LRP1 reduces the levels of both PIP5KL1 and PIP5K1ß in the plasma membrane and also lowers phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) levels in hepatocytes. These data indicate that LRP1 recruits PIP5KL1 and PIP5K1ß to the plasma membrane for PI(4,5)P2 biosynthesis. The lack of LRP1 reduces lipid kinase expression, leading to lower PI(4,5)P2 levels, thereby decreasing the availability of this lipid metabolite in the cardiolipin biosynthesis pathway to cause cardiolipin reduction and the impairment in mitochondria homeostasis. Taken together, the current study identifies another signaling mechanism by which LRP1 regulates cell functions: binding and recruitment of PIP5KL1 and PIP5K1ß to the membrane for PI(4,5)P2 synthesis. In addition, it highlights the importance of this mechanism for maintaining the integrity and functions of intracellular organelles.

Low-density lipoprotein receptor-related protein 1 deficiency in cardiomyocytes reduces susceptibility to insulin resistance and obesity.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP1) plays a key role in fatty acid metabolism and glucose homeostasis. In the context of dyslipemia, LRP1 is upregulated in the heart. Our aim was to evaluate the impact of cardiomyocyte LRP1 deficiency on high fat diet (HFD)-induced cardiac and metabolic alterations, and to explore the potential mechanisms involved. METHODS: We used TnT-iCre transgenic mice with thoroughly tested suitability to delete genes exclusively in cardiomyocytes to generate an experimental mouse model with conditional Lrp1 deficiency in cardiomyocytes (TNT-iCre+-LRP1flox/flox). FINDINGS: Mice with Lrp1-deficient cardiomyocytes (cm-Lrp1-/-) have a normal cardiac function combined with a favorable metabolic phenotype against HFD-induced glucose intolerance and obesity. Glucose intolerance protection was linked to higher hepatic fatty acid oxidation (FAO), lower liver steatosis and increased whole-body energy expenditure. Proteomic studies of the heart revealed decreased levels of cardiac pro-atrial natriuretic peptide (pro-ANP), which was parallel to higher ANP circulating levels. cm-Lrp1-/- mice showed ANP signaling activation that was linked to increased fatty acid (FA) uptake and increased AMPK/ ACC phosphorylation in the liver. Natriuretic peptide receptor A (NPR-A) antagonist completely abolished ANP signaling and metabolic protection in cm-Lrp1-/- mice. CONCLUSIONS: These results indicate that an ANP-dependent axis controlled by cardiac LRP1 levels modulates AMPK activity in the liver, energy homeostasis and whole-body metabolism.

LRP1 Deficiency in Vascular SMC Leads to Pulmonary Arterial Hypertension That Is Reversed by PPARγ Activation.

RATIONALE: Arterial remodeling-a hallmark of many cardiovascular pathologies including pulmonary arterial hypertension (PAH)-is regulated by TGFß1 (transforming growth factor-ß1)-TGFß receptors and the antagonistic, vasoprotective BMPR2 (bone morphogenetic protein receptor 2)-PPARγ (peroxisome proliferator-activated receptor-γ) axis. However, it is unclear which factors drive detrimental TGFß1 pathways in the hypertensive pulmonary vasculature. OBJECTIVE: We hypothesized that LRP1 (low-density lipoprotein receptor-related protein 1) expression is decreased in PAH, leading to enhancement (disinhibition) of TGFß1 signals and that the PPARγ agonist pioglitazone can restore vascular homeostasis and prevent PAH resulting from LRP1 deletion in vascular smooth muscle cells (SMCs). METHODS AND RESULTS: Targeted deletion of LRP1 in vascular SMC (smLRP1-/-) in mice disinhibited TGFß1-CTGF (connective tissue growth factor) signaling, leading to spontaneous PAH and distal pulmonary arterial muscularization as assessed by closed-chest cardiac catheterization and anti-αSMA staining. Pioglitazone inhibited the canonical TGFß1-CTGF axis in human pulmonary artery SMC and smLRP1-/- main pulmonary artery (CTGF and NOX4) and reversed PAH in smLRP1-/- mice. TGFß1 boosted pSmad3 in PASMC from smLRP1-/- mice versus controls. Pioglitazone-activated PPARγ binds to Smad3 in human pulmonary artery SMC (coimmunoprecipitation), thereby blocking its phosphorylation and overriding LRP1 deficiency. Finally, mRNA and protein expression of LRP1 was decreased in pulmonary plexiform lesions of patients with end-stage idiopathic PAH (laser capture microdissection, qPCR, and immunohistochemistry). Downregulation of LRP1 protein was also demonstrated in explanted PASMC from patients with PAH and accompanied by enhanced TGFß1-pSmad3-CTGF signaling and increased TGFß1-induced PASMC proliferation that was prevented by pioglitazone. CONCLUSIONS: Here, we identify LRP1 as an integrator of TGFß1-mediated mechanisms that regulate vascular remodeling in mice and clinical PAH and PPARγ as a therapeutic target that controls canonical TGFß1 pathways. Hence, pharmacologic PPARγ activation represents a promising new therapy for patients with PAH who lack the vasoprotective LRP1 in vascular SMC.

LRP1 deficiency in microglia blocks neuro-inflammation in the spinal dorsal horn and neuropathic pain processing.

Following injury to the peripheral nervous system (PNS), microglia in the spinal dorsal horn (SDH) become activated and contribute to the development of local neuro-inflammation, which may regulate neuropathic pain processing. The molecular mechanisms that control microglial activation and its effects on neuropathic pain remain incompletely understood. We deleted the gene encoding the plasma membrane receptor, LDL Receptor-related Protein-1 (LRP1), conditionally in microglia using two distinct promoter-Cre recombinase systems in mice. LRP1 deletion in microglia blocked development of tactile allodynia, a neuropathic pain-related behavior, after partial sciatic nerve ligation (PNL). LRP1 deletion also substantially attenuated microglial activation and pro-inflammatory cytokine expression in the SDH following PNL. Because LRP1 shedding from microglial plasma membranes generates a highly pro-inflammatory soluble product, we demonstrated that factors which activate spinal cord microglia, including lipopolysaccharide (LPS) and colony-stimulating factor-1, promote LRP1 shedding. Proteinases known to mediate LRP1 shedding, including ADAM10 and ADAM17, were expressed at increased levels in the SDH after PNL. Furthermore, LRP1-deficient microglia in cell culture expressed significantly decreased levels of interleukin-1ß and interleukin-6 when treated with LPS. We conclude that in the SDH, microglial LRP1 plays an important role in establishing and/or amplifying local neuro-inflammation and neuropathic pain following PNS injury. The responsible mechanism most likely involves proteolytic release of LRP1 from the plasma membrane to generate a soluble product that functions similarly to pro-inflammatory cytokines in mediating crosstalk between cells in the SDH and in regulating neuropathic pain.

Methotrexate and its therapeutic antagonists caffeine and theophylline, target a motogenic T-cell mechanism driven by thrombospondin-1 (TSP-1).

Methotrexate (MTX) is a widely used treatment for inflammatory diseases such as rheumatoid arthritis and psoriasis, based on the concept that it is immunosuppressive. Its mechanism of action, however, remains unclear, although it is thought to depend on adenosine. Caffeine and theophylline, which have several targets including adenosine receptors, have been shown to suppress the beneficial clinical effects of MTX. Here we show that MTX and caffeine and theophylline differentially affect a motogenic T-cell mechanism driven by endogenous thrombospondin-1 (TSP-1) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1). MTX stimulated TSP-1 expression and the motogenic TSP-1/TSP-1 receptor mechanism in primary human T cells, hence mimicking IL-2 and CXCL12, which similar to MTX, dampen inflammatory disease. SiRNA-mediated gene silencing of TSP-1 and LRP1 inhibited this stimulatory effect. Caffeine and theophylline inhibited the TSP-1/TSP-1 receptor mechanism by inhibiting LRP1 expression. These results indicate that the effect of MTX on T cells is immunoregulatory rather than immunosuppressive, and suggest a pathway dependent on TSP-1/TSP-1 receptor interactions for the regulation of immune responses.

What are lipoproteins doing in the brain?

Lipoproteins in plasma transport lipids between tissues, however, only high-density lipoproteins (HDL) appear to traverse the blood-brain barrier (BBB); thus, lipoproteins found in the brain must be produced within the central nervous system. Apolipoproteins E (ApoE) and ApoJ are the most abundant apolipoproteins in the brain, are mostly synthesized by astrocytes, and are found on HDL. In the hippocampus and other brain regions, lipoproteins help to regulate neurobehavioral functions by processes that are lipoprotein receptor-mediated. Moreover, lipoproteins and their receptors also have roles in the regulation of body weight and energy balance, acting through lipoprotein lipase (LPL) and the low-density lipoprotein (LDL) receptor-related protein (LRP). Thus, understanding lipoproteins and their metabolism in the brain provides a new opportunity with potential therapeutic relevance.

Macrophage LRP1 suppresses neo-intima formation during vascular remodeling by modulating the TGF-ß signaling pathway.

BACKGROUND: Vascular remodeling in response to alterations in blood flow has been shown to modulate the formation of neo-intima. This process results from a proliferative response of vascular smooth muscle cells and is influenced by macrophages, which potentiate the development of the intima. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that recognizes a number of ligands including apoE-containing lipoproteins, proteases and protease-inhibitor complexes. Macrophage LRP1 is known to influence the development of atherosclerosis, but its role in vascular remodeling has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: To define the contribution of macrophage LRP1 to vascular remodeling, we generated macrophage specific LRP1-deficient mice (macLRP1-/-) on an LDL receptor (LDLr) knock-out background. Using a carotid ligation model, we detected a 2-fold increase in neointimal thickening and a 2-fold increase in the intima/media ratio in macLRP1-/- mice. Quantitative RT-PCR arrays of the remodeled vessel wall identified increases in mRNA levels of the TGF-ß2 gene as well as the Pdgfa gene in macLRP1-/- mice which could account for the alterations in vascular remodeling. Immunohistochemistry analysis revealed increased activation of the TGF-ß signaling pathway in macLRP1-/- mice. Further, we observed that LRP1 binds TGF-ß2 and macrophages lacking LRP1 accumulate twice as much TGF-ß2 in conditioned media. Finally, TNF-α modulation of the TGF-ß2 gene in macrophages is attenuated when LRP1 is expressed. Together, the data reveal that LRP1 modulates both the expression and protein levels of TGF-ß2 in macrophages. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that macrophage LRP1 protects the vasculature by limiting remodeling events associated with flow. This appears to occur by the ability of macrophage LRP1 to reduce TGF-ß2 protein levels and to attenuate expression of the TGF-ß2 gene resulting in suppression of the TGF-ß signaling pathway.

Lrp12/Mig13a reveals changing patterns of preplate neuronal polarity during corticogenesis that are absent in reeler mutant mice.

During corticogenesis, the earliest generated neurons form the preplate, which evolves into the marginal zone and subplate. Lrp12/Mig13a, a mammalian gene related to the Caenorhabditis elegans neuroblast migration gene mig-13, is expressed in a subpopulation of preplate neurons that undergo ventrally directed tangential migrations in the preplate layer and pioneer axon projections to the anterior commissure. As the preplate separates, Lrp12/Mig13a-positive neurons polarize in the radial plane and form a pseudocolumnar pattern, prior to moving to a deeper position within the emerging subplate layer. These changes in neuronal polarity do not occur in reeler mutant mice, revealing the earliest known defect in reeler cortical patterning and suggesting that the alignment of preplate neurons into a pseudolayer facilitates the movement of later-born radially migrating neurons into the emerging cortical plate.

Downregulation of functional Reelin receptors in projection neurons implies that primary Reelin action occurs at early/premigratory stages.

Reelin signaling is essential for correct development of the mammalian brain. Reelin binds to apolipoprotein E receptor 2 and very low-density lipoprotein receptor and induces phosphorylation of Dab1. However, when and where these reactions occur is essentially unknown, and the primary function(s) of Reelin remain unclear. Here, we used alkaline phosphatase fusion of the receptor-binding region of Reelin to quantitatively investigate the localization of functional Reelin receptors (i.e., those on the plasma membrane as mature forms) in the developing brain. In the wild-type cerebral cortex, they are mainly present in the intermediate and subventricular zones, as well as in radial fibers, but much less in the cell bodies of the cortical plate. Functional Reelin receptors are much more abundant in the Reelin-deficient cortical plate, indicating that Reelin induces their downregulation and that it begins before the neurons migrate out of the intermediate zone. In the wild-type cerebellum, functional Reelin receptors are mainly present in the cerebellar ventricular zone but scarcely expressed by Purkinje cells that have migrated out of it. It is thus strongly suggested that Reelin exerts critical actions on migrating projection neurons at their early/premigratory stages en route to their final destinations, in the developing cerebral cortex and cerebellum.

Peptide-mediated targeting of hepatocytes via low density lipoprotein receptor-related protein (LRP).

It has previously been reported that a peptide sequence of T7 phage protein p17 mediates uptake of its cargo by liver parenchymal cells. The aim of this study was to identify the phage-binding receptor. The involvement of LRP was confirmed by the observations that phage binding to Hepa 1c1c7 cells was inhibited by the LRP-binding receptor-associated protein, LRP-deficient mouse embryonic fibroblasts bound phage with lower efficiency than their wild-type counterparts, and using mouse models with ablated LRP liver expression. The identification of LRP as a cognate receptor for this sequence offers a new ligand-receptor combination for hepatocyte delivery of therapeutic agents.

Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis.

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.

The low-density lipoprotein receptor-related protein regulates cancer cell survival and metastasis development.

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. In this study, we show that LRP-1 is abundantly expressed in severe combined immunodeficient (SCID) mouse xenografts by various human cancer cell lines that express very low or undetectable levels of LRP-1 when cultured in 21% O2 in vitro (standard cell culture conditions). To test whether LRP-1 expression in vivo may be explained by hypoxia in the xenografts, CL16 cells, which are derived from the MDA-MB-435 cell line, were cultured in 1.0% O2. A substantial increase in LRP-1 expression was observed. To test the activity of LRP-1 in cancer progression in vivo, LRP-1 expression was silenced in CL16 cells with short hairpin RNA. These cells formed tumors in SCID mice, in which LRP-1 expression remained silenced. Although LRP-1 gene silencing did not inhibit CL16 cell dissemination from the primary tumors to the lungs, the pulmonary metastases failed to enlarge, suggesting compromised survival or growth at the implantation site. In cell culture experiments, significantly increased cell death was observed when LRP-1-silenced CL16 cells were exposed to CoCl2, which models changes that occur in hypoxia. Furthermore, LRP-1-silenced cells expressed decreased levels of vascular endothelial growth factor in response to 1.0% O2. These results suggest mechanisms by which LRP-1 may facilitate the development and growth of cancer metastases in vivo.

Deletion of macrophage LDL receptor-related protein increases atherogenesis in the mouse.

Macrophage low-density lipoprotein receptor-related protein (LRP) mediates internalization of remnant lipoproteins, and it is generally thought that blocking lipoprotein internalization will reduce foam cell formation and atherogenesis. Therefore, our study examined the function of macrophage LRP in atherogenesis. We generated transgenic mice that specifically lack macrophage LRP through Cre/lox recombination. Transplantation of macrophage LRP(-/-) bone marrow into lethally irradiated female LDLR(-/-) recipient mice resulted in a 40% increase in atherosclerosis. The difference in atherosclerosis was not caused by altered serum lipoprotein levels. Furthermore, deletion of macrophage LRP decreased uptake of (125)I-very-low-density lipoprotein compared with wild-type cells in vitro. The increase in atherosclerosis was accompanied by increases in monocyte chemoattractant protein type-1, tumor necrosis factor-alpha, and proximal aorta macrophage cellularity. We also found that deletion of macrophage LRP increases matrix metalloproteinase-9. This increase in matrix metalloproteinase-9 was associated with a higher frequency of breaks in the elastic lamina. Contrary to what was found with other lipoprotein receptors, deletion of LRP increases atherogenesis in hypercholesterolemic mice. Our data support the hypothesis that macrophage LRP modulates atherogenesis through regulation of inflammatory responses.

Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor beta (PDGFR beta).

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.

FE65 constitutes the functional link between the low-density lipoprotein receptor-related protein and the amyloid precursor protein.

Increasing evidence has implicated the low density lipoprotein receptor-related protein (LRP) and the adaptor protein FE65 in Alzheimer's disease pathogenesis. We have shown previously that LRP mediates beta-amyloid precursor protein (APP) processing and affects amyloid beta-protein and APP secretion and APP-c-terminal fragment generation. Furthermore, LRP mediates APP processing through its intracellular domain. Here, we set out to examine whether this interaction is of direct or indirect nature. Specifically, we asked whether adaptor proteins such as FE65 influence the LRP-mediated effect on APP processing by forming a protein complex. In coimmunoprecipitation experiments, we confirmed the postulated APP-FE65 and the LRP-FE65 interaction. However, we also showed an LRP-FE65-APP trimeric complex using pull-down techniques. Because FE65 alters APP processing, we investigated whether this effect is LRP dependent. Indeed, FE65 was only able to increase APP secretion in the presence of LRP. In the absence of LRP, APP secretion was unchanged compared with the LRP knock-out phenotype. Using RNA short interference techniques against FE65, we demonstrated that a reduction in FE65 protein mimics the LRP knock-out phenotype on APP processing. These results clearly demonstrate that FE65 acts as a functional linker between APP and LRP.

Hepatic low-density lipoprotein receptor-related protein deficiency in mice increases atherosclerosis independent of plasma cholesterol.

The low-density lipoprotein (LDL) receptor-related protein (LRP) has a well-established role in the hepatic removal of atherogenic apolipoprotein E (APOE)-rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and antiatherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double-deficient mice (MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-)). On an LDLR(-/-)APOE(-/-) background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1 +/- 5.2 vs 23.4 +/- 6.3 mM, P =.025; triglycerides: 1.1 +/- 0.5 vs 2.2 +/- 0.8 mM, P =.002, for MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) and control LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice, respectively). Lower plasma cholesterol in MX1Cre(+)LRP(flox/flox)-LDLR(-/-)APOE(-/-) mice coincided with increased plasma lipoprotein lipase (71.2 +/- 7.5 vs 19.1 +/- 2.4 ng/ml, P =.002), coagulation factor VIII (4.4 +/- 1.1 vs 1.9 +/- 0.5 U/mL, P =.001), von Willebrand factor (2.8 +/- 0.6 vs 1.4 +/- 0.3 U/mL, P =.001), and tissue-type plasminogen activator (1.7 +/- 0.7 vs 0.9 +/- 0.5 ng/ml, P =.008) compared with controls. Strikingly, MX1Cre(+)LRP(flox/flox)LDLR(-/-)APOE(-/-) mice showed a 2-fold higher atherosclerotic lesion area compared with controls (408.5 +/- 115.1 vs 219.1 +/- 86.0 10(3)microm(2), P =.003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its proatherogenic ligands.